Abstract Hemophilia A is the most common severe congenital bleeding disorder and is the result of a deficiency of the clotting factor (F) VIII. Clinical severity is largely determined by a patient's baseline factor level: severe (<1%), moderate (1-5%) and mild (5-30%), but it is well described that 10-15% of patients with severe factor VIII deficiency have a mild bleeding phenotype. The biologic factors that underlie this variability remain poorly understood. This proposal will be investigating a novel mechanism of phenotypic variability in hemophilia A. In the plasma, FVIII is bound to von Willebrand factor (VWF). VWF is essential for platelet adhesion at the site of vascular injury and thrombus formation. VWF is synthesized in endothelial cells and secreted into the blood stream as ultra large (UL) multimers. Proteolytic cleavage of these UL-VWF multimers by ADAMTS13 metalloprotease decreases VWF function. The persistence of UL-VWF multimers secondary to decreased proteolysis by ADAMTS13 results in a prothrombotic state. The applicant's co-mentor has demonstrated in murine studies that the presence of FVIII accelerates the cleavage of VWF by ADAMTS13 under fluid shear stress. Without binding of FVIII to VWF, there is decreased VWF cleavage and increased multimeric size. I hypothesize that this observation of the interaction of FVIII, ADAMTS13 and VWF underlies, in part, the heterogeneity observed in bleeding manifestations in patients with severe FVIII deficiency. Severe hemophilia A patients who have no circulating FVIII protein will have increased UL-VWF multimers and a milder clinical bleeding phenotype than those with circulating, but inactive, FVIII. We will address this hypothesis through the following specific aims: 1) determine the VWF multimer pattern in patients with hemophilia A compared to healthy controls and correlate multimer size with the presence or absence of circulating FVIII 2) assess in a retrospective cohort study the association of UL-VWF multimers and a mild clinical bleeding phenotype, and 3) examine in a prospective intervention study the dynamic change in the VWF multimer pattern with the infusion of recombinant FVIII. This proposed study will shed new light on the role of FVIII in VWF processing by ADAMTS13 and on understanding phenotypic variability in patients with severe hemophilia A. Distinction of phenotype in young patients with hemophilia A prior to the onset of bleeding will improve prognosis and allow for individualization of treatment. Also the discovery of a novel mechanism that modifies severity of bleeding in hemophilia may offer new therapeutic strategies. The applicant is a pediatric hematologist with interest in clinical research in hemophilia. She has published several first authored, original papers related to complications in hemophilia and their therapies, and is completing a masters in clinical epidemiology. The proposed mentoring program allows her to consolidate her formal clinical coursework training and gain mentored experience in a highly innovative translational research plan that is directly applicable to the care of patients with hemophilia. This proposed mentored experience will take place in one of the most outstanding hemostasis and thrombosis research to clinical environments in the country as well. The proposed mentoring experience will be directed by an experienced physician-scientist in the hemostasis and thrombosis field, Dr. Poncz, with an outstanding reputation in supporting the development of junior faculty into independent, NIH-funded, clinical researchers.